Eukaryotic expression: developments for structural proteomics

The production of sufficient quantities of protein is an essential prelude to a structure determination, but for many viral and human proteins this cannot be achieved using prokaryotic expression systems. Groups in the Structural Proteomics In Europe ( SPINE) consortium have developed and implemented high- throughput ( HTP) methodologies for cloning, expression screening and protein production in eukaryotic systems. Studies focused on three systems: yeast ( Pichia pastoris and Saccharomyces cerevisiae), baculovirusinfected insect cells and transient expression in mammalian cells. Suitable vectors for HTP cloning are described and results from their use in expression screening and protein-production pipelines are reported. Strategies for coexpression, selenomethionine labelling ( in all three eukaryotic systems) and control of glycosylation ( for secreted proteins in mammalian cells) are assessed.

Mechanical effects of plant cell wall enzymes on xyloglucan/cellulose composites

Xyloglucan-acting enzymes are believed to have effects on type I primary plant cell wall mechanical properties. In order to get a better understanding of these effects, a range of enzymes with different in vitro modes of action were tested against cell wall analogues (bio-composite materials based on Acetobacter xylinus cellulose and xyloglucan). Tomato pericarp xyloglucan endo transglycosylase (tXET) and nasturtium seed xyloglucanase (nXGase) were produced heterologously in Pichia pastoris. Their action against the cell wall analogues was compared with that of a commercial preparation of Trichoderma endo-glucanase (EndoGase). Both 'hydrolytic' enzymes (nXGase and EndoGase) were able to depolymerise not only the cross-link xyloglucan fraction but also the surface-bound fraction. Consequent major changes in cellulose fibril architecture were observed. In mechanical terms, removal of xyloglucan cross-links from composites resulted in increased stiffness (at high strain) and decreased visco-elasticity with similar extensibility. On the other hand, true transglycosylase activity (tXET) did not affect the cellulose/xyloglucan ratio. No change in composite stiffness or extensibility resulted, but a significant increase in creep behaviour was observed in the presence of active tXET. These results provide direct in vitro evidence for the involvement of cell wall xyloglucan-specific enzymes in mechanical changes underlying plant cell wall re-modelling and growth processes. Mechanical consequences of tXET action are shown to be complimentary to those of cucumber expansin.

Claviceps purpurea expressing polygalacturonases escaping PGIP inhibition fully infects PvPGIP2 wheat transgenic plants but its infection is delayed in wheat transgenic plants with increased level of pectin methyl esterification

Claviceps purpurea is a biotrophic fungal pathogen of grasses causing the ergot disease. The infection process of C. purpurea on rye flowers is accompanied by pectin degradation and polygalacturonase (PG) activity represents a pathogenicity factor. Wheat is also infected by C. purpurea and we tested whether the presence of polygalacturonase inhibiting protein (PGIP) can affect pathogen infection and ergot disease development. Wheat transgenic plants expressing the bean PvPGIP2 did not show a clear reduction of disease symptoms when infected with C. purpurea. To ascertain the possible cause underlying this lack of improved resistance of PvPGIP2 plants, we expressed both polygalacturonases present in the C. purpurea genome, cppg1 and cppg2 in Pichia pastoris. In vitro assays using the heterologous expressed PGs and PvPGIP2 showed that neither PG is inhibited by this inhibitor. To further investigate the role of PG in the C. purpurea/wheat system, we demonstrated that the activity of both PGs of C. purpurea is reduced on highly methyl esterified pectin. Finally, we showed that this reduction in PG activity is relevant in planta, by inoculating with C. purpurea transgenic wheat plants overexpressing a pectin methyl esterase inhibitor (PMEI) and showing a high degree of pectin methyl esterification. We observed reduced disease symptoms in the transgenic line compared with null controls. Together, these results highlight the importance of pectin degradation for ergot disease development in wheat and sustain the notion that inhibition of pectin degradation may represent a possible route to control of ergot in cereals.

Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of similar to 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80-150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 degrees C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 degrees C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K(M) was 42 mM, and V(max) was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence. (C) 2008 Elsevier Inc. All rights reserved.

Structure, processing and midgut secretion of putative peritrophic membrane ancillary protein (PMAP) from Tenebrio molitor larvae

A cDNA coding for a Tenebrio molitor midgut protein named peritrophic membrane ancillary protein (PMAP) was cloned and sequenced. The complete cDNA codes for a protein of 595 amino acids with six insect-allergen-related-repeats that may be grouped in A (predicted globular)- and B (predicted nonglobular)-types forming an ABABAB structure. The PMAP-cDNA was expressed in Pichia pastoris and the recombinant protein (64 kDa) was purified to homogeneity and used to raise antibodies in rabbits. The specific antibody detected PMAP peptides (22 kDa) in the anterior and middle midgut tissue, luminal contents, peritrophic membrane and feces. These peptides derive from PMAP, as supported by mass spectrometry, and resemble those formed by the in vitro action of trypsin on recombinant PMAP. Both in vitro and in vivo PMAP processing seem to occur by attack of trypsin to susceptible bonds in the coils predicted to link AB pairs, thus releasing the putative functional AB structures. The AB-domain structure of PMAP is found in homologous proteins from several insect orders, except lepidopterans that have the apparently derived protein known as nitrile-specifier protein. Immunocytolocalization shows that PMAP is secreted by exocytosis and becomes entrapped in the glycocalyx, before being released into midgut contents. Circumstantial evidence suggests that PMAP-like proteins have a role in peritrophic membrane type 2 formation. (C) 2007 Elsevier Ltd. All rights reserved.

Avaliação da atividade biológica de óleos essenciais sobre candida não albicans de origem clínica

Candidíase é um problema de importância crescente, devido o aumento do número de indivíduos imunocomprometidos e o surgimento de cepas resistentes aos antifúngicos convencionais. É de fundamental importância a busca por novos agentes antifúngicos mais eficazes, menos tóxicos, sendo os óleos essenciais (OEs) excelentes alternativas para esse propósito. Esse estudo investigou a atividade biológica do OE de Mentha spicata L. sobre Candida guilliermondii de origem anal e vaginal. Para tanto foram determinadas a Concentração Inibitória Mínima (CIM), Concentração Fungicida Mínima (CFM), cinética do crescimento das leveduras (Time-Kill), alterações micromorfológicas (técnica do microcultivo em câmara úmida) e investigação do mecanismo de ação antifúngico, utilizando o bioensaio do sorbitol. O OE de M. spicata foi obtido pelo processo de extração por destilação a vapor. Na análise fitoquímica desse óleo foi observada a presença de carvona com 84,32%, seguida pelo limoneno (13,70%) e traços de iso-dihidrocarvona (0,82%). Os resultados da análise da CIM variou entre 32 e 128 μg/mL. A CFM variou entre 64 e 1024 μg/mL. Na avaliação da ação de OE e da nistatina 100UI/mL, o antifúngico padrão apresentou o efeito fungicida a partir de 4 horas e para OE de M. spicata foi observado efeito fungistático na CIM, CIMX2 e CIMX4 frente às cepas avaliadas. O OE de M. spicata apresentou forte atividade antifúngica contra as cepas de C. guilliermondii, promovendo alterações micromorfológicas visíveis por microscopia óptica, nas concentrações testadas (CIM, CIMx2), resultado semelhante ao que se observou com a nistatina (100UI/mL). Na investigação do mecanismo de ação antifúngico foi constatado que houve alteração da CIM na presença de sorbitol, com elevação dos valores quatro vezes maior que a concentração inicial, o que indica que os componentes desse OE apresentam ação direta sobre a parede celular das leveduras. Conclui-se que o OE de Mentha spicata é um potencial agente terapêutico no tratamento de candidíase

Avaliação do sistema conservante em formulação com extrato hidroalcóolico de schinus terebinthifolius raddi nacardiaceae

Schinus terebinthifolius Raddi is used in the treatment of skin and mucosal injuries, infections of respiratory, digestive and genitourinary systems. Currently one of the biggest problems faced for the industry of phytopharmaceuticals with regard to the quality of raw materials is the microbial contamination. The aim this study was to evaluate the antimicrobial action of the hidroalcoholic extract of aroeira, beyond testing the effectiveness of the preservative system in hidrogel to the base of this extract. The extracts were prepared by maceration in the ratio of 1:10 of solvent plant/with alcohol 40%. The methods for microbial count were pour plate and test for specific microorganisms, analyzing in third copy each one of the samples. The antimicrobial activity of aroeira extracts was performed using an agar diffusion method, using strains of S. aureus, P. aeruginosa, E. coli, B. subtilis, C. albicans, C. tropicalis, C. krusei, C. guilliermondii, T. rubrum, M. gypseum, A. flavus and A. niger. The formula with aroeira was evaluated by the challenge test. This method consisted of artificial contamination the sample with separate inóculos of A. niger, C. albicans, E. coli e S. aureus aeruginosa and determinations of survivors for the method of counting for pour plate , during times 0, 24h, 48h, 7 days, 14 days, 21 days and 28 days. How much to the results, one verified that the extract of aroeira in the 13,5 concentration mg/mL presented antimicrobial activity for cepas of E. coli, B. subtilis, P. aeruginosa e S. aureus, producing inhibition zone, on average with 13 mm of diameter. However it did not present no fungi activity. The formula with aroeira containig both methylparaben and propylparaben showed a good efficacy in challenge test front to strains of A. niger, C. albicans, E. coli, S. aureus. The A criteria of European Pharmacopoeia, adopted in this work, was verified that this product revealed the good preservative efficacy for the challenge test, time interval of the 28 days. However, it is interesting to extend this study, in order to carry through the sped up stability and the test of shelf, to establish the validity of this formularization

Conditions affecting production of functional muscle recombinant alpha-tropomyosin in Saccharomyces cerevisiae

Yeasts are attractive hosts for heterologous protein production as they follow the general eukaryotic post-translational modification pattern. The well-known Saccharomyces cerevisiae has been used to produce a large variety of foreign proteins. The proper function of muscle tropomyosin depends on a specific modification at its N-terminus. Although tropomyosin has been produced in different expression systems, only the recombinant protein produced in the yeast Pichia pastoris has native-like functional properties. In this paper we describe the production of functional skeletal muscle tropomyosin in the yeast S. cerevisiae. The recombinant protein was produced in high amounts and production was strongly affected by genetic and environmental factors, including plasmid copy number, promoter strength, and growth media composition. (C) 2003 Elsevier B.V. (USA). All rights reserved.

Monitoramento de fungos anemófilos e de leveduras em unidade hospitalar

OBJETIVO: Monitorar e caracterizar fungos anemófilos e leveduras de fontes bióticas e abióticas de uma unidade hospitalar. MÉTODOS: As coletas foram realizadas mensalmente e em dois períodos, do centro cirúrgico e unidades de terapia intensiva adulto e neonatal em hospital de Araraquara, Estado de São Paulo. Para coleta de fungos anemófilos foi utilizado amostrador tipo Andersen de simples estágio. A pesquisa de leveduras foi feita das mãos e de orofaringe de profissionais de saúde, bem como de superfícies de leitos e de maçanetas das áreas críticas. RESULTADOS: Foram recuperados do centro cirúrgico 32 gêneros de fungos anemófilos e 31 das unidades de terapia intensiva. Os gêneros mais freqüentemente isolados foram Cladophialophora spp., Fusarium spp., Penicillium spp., Chrysosporium spp. e Aspergillus spp. Durante o período de estudo, houve reforma e implantação de uma unidade dentro do hospital, que coincidiu com o aumento na contagem de colônias de Cladophialophora spp., Aspergillus spp. e Fusarium spp. Leveduras foram encontradas em 39,4% dos profissionais de saúde (16,7% das amostras dos espaços interdigitais, 12,1% do leito subungueal e 10,6% da orofaringe) e, em 44% das amostras do mobiliário, com predomínio do gênero Candida (C. albicans, C. guilliermondii, C. parapsilosis e C. lusitaniae) seguido por Trichosporon spp. CONCLUSÕES: Observou-se número relativamente elevado de fungos anemófilos (potencialmente patogênicos) em áreas especiais e níveis expressivos de leveduras em fontes bióticas e abióticas. O monitoramento microbiológico ambiental deve ser realizado, principalmente em salas especiais com pacientes imunocomprometidos, sujeitos à exposição de patógenos do meio ambiente, assim como, advindos de profissionais de saúde.

Genetic relatedness of commensal strains of Candida albicans carried in the oral cavity of patients' dental prosthesis users in Brazil

The aim of this study is to describe the degree of yeast-colonization in diabetic and hemodialysed-users of dental prostheses. Individuals (306) were examined using an oral rinse technique in order to evaluate the incidence of yeast-carriage, and genotype of C. albicans. Yeasts were isolated from 68.4% (91/133) individual's dental prostheses users. Dental prostheses were found to be a significant factor for the yeast colonization (P < 0.05). Overall, the intensity of carriage was higher in diabetic patients as compared with health and hemodialysed individuals (P < 0.05). The isolation rates were: C. albicans (51.7%), C. parapsilosis (20.9%), C. tropicalis (14.3%), C. glabrata (6.6%), C. krusei (3.3%), C. rugosa (1.1%), and Pichia (Pichia ohmeri, 2.2%). Ready-To-Go RAPD Analysis Beads were used and primer OPJ 6 distinguished the C. albicans isolates found in prostheses users. All the isolates were grouped into 11 RAPD profiles in four main clusters and, the average S (AB) for the entire collection of 47 C. albicans isolates were 0.779 +/- 0.178. Over 85% of isolates had a similarity level higher than or equal to 0.8 reinforcing the idea that the use of dental prostheses, independently of the host's clinical condition, probably provides the necessary conditions for these strains to gain a growth-specific advantage over others.

Candida spp.: manual identification (reference method) and automated identification (Vitek system platform)

Yeasts are becoming a common cause of nosocomial fungal infections that affect immunocompromised patients. Such infections can evolve into sepsis, whose mortality rate is high. This study aimed to evaluate the viability of Candida species identification by the automated system Vitek-Biomerieux (Durham, USA). Ninety-eight medical charts referencing the Candida spp. samples available for the study were retrospectively analyzed. The system Vitek-Biomerieux with Candida identification card is recommended for laboratory routine use and presents 80.6% agreement with the reference method. By separate analysis of species, 13.5% of C. parapsilosis samples differed from the reference method, while the Vitek system wrongly identified them as C. tropicalis, C. lusitaneae or as Candida albicans. C. glabrata presented a discrepancy of only one sample (25%), and was identified by Vitek as C. parapsilosis. C. guilliermondii also differed in only one sample (33.3%), being identified as Candida spp. All C. albicans, C. tropicalis and C. lusitaneae samples were identified correctly.

Road-killed wild animals: a preservation problem useful for eco-epidemiological studies of pathogens

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Cross-reactive IgE antibody responses to tropomyosins from Ascaris lumbricoides and cockroach

Background: Evidence indicates that infection with Ascaris lumbricoides may promote development of allergy and asthma.Objective: To study the role of tropomyosin, a pan-allergen in invertebrates, in IgE responses to A lumbricoides.Methods: Recombinant A lumbricoides and Periplaneta americana tropomyosins were expressed in Pichia pastoris. Levels of IgE to tropomyosins from A lumbricoides and P americana were determined by chimeric ELISA in sera from 119 children living in a parasite-endemic area and 112 patients with cockroach allergy from the allergy clinics. Presence of tropomyosin in A lumbricoides larvae at L3 stage was evaluated by immunofluorescence using mAb IA6, directed against mite tropomyosin. Molecular modeling of P americana and A lumbricoides tropomyosins was performed by using the MODELLER program.Results: A lumbricoides tropomyosin showed 69% to 98% sequence identity to tropomyosins from other invertebrates. The predicted structure of A lumbricoides tropomyosin was similar to that of P americana tropomyosin and showed the characteristic coiled-coil structure. Strong correlation was found for IgE antibodies to tropomyosins from A lumbricoides and P americana in sera from children living in a parasite-endemic area and from patients with cockroach allergy. Larvae of A lumbricoides reacted strongly with mAb IA6.Conclusion: Tropomyosin induces IgE responses in A lumbricoides-infected children and in patients allergic to cockroach.

Candida heliconiae sp nov., Candida picinguabensis sp nov and Candida saopaulonensis sp nov., three ascomycetous yeasts from Heliconia velloziana (Heliconiaceae)

Strains belonging to three novel yeast species, Candida heliconiae (four isolates), Candida picinguabensis (three isolates) and Candida saopaulonensis (two isolates), were recovered in the year 2000 from water of flower bracts of Heliconia velloziana L. Emigd. (Heliconiaceae) found in a forest ecosystem site in an Atlantic rainforest of south-eastern Brazil. C. picinguabensis and C. saopaulonensis were nearly identical in morphology and physiology, but sequence divergence in the D1/D2 domain of the large-subunit rDNA indicated that they should be regarded as different species. They belong to the Metschnikowiaceae clade. C. heliconiae had affinities to Pichia mexicana and related species, but was genetically isolated from all currently accepted species in that group. The type strains are C. heliconiae UNESP 00-91 C1(T) (= CBS 10000(T) = NRRL Y-27813(T)), C. picinguabensis UNESP 00-89(T) (= CBS 9999(T) = NRRL Y-27814(T)) and C. saopaulonensis UNESP 00-99(T) (=CBS 10001(T) = NRRL Y-27815(T)).

Culture-dependent and culture-independent characterization of microorganisms associated with Aedes aegypti (Diptera: Culicidae) (L.) and dynamics of bacterial colonization in the midgut

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)